|
| Status |
Public on Nov 27, 2014 |
| Title |
Lactation day 87 [TR05_110903_W685] |
| Sample type |
RNA |
| |
|
| Source name |
Suckled mammary gland
|
| Organism |
Notamacropus eugenii |
| Characteristics |
day lactation: Lactation day 87
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
1. Total RNA was isolated from mammary gland tissue by purification through a silica column using an RNeasy Midi kit (QIAGEN) as per manufacture's instruction (QIAGEN)
2. Subsequent mRNA was extracted from total RNA with poly T oligomers attached to superparamagnetic beads using the DynaBeads mRNA Purification Kit (Dynal Biotech) as per manufacture's instructions
|
| Label |
Cy5
|
| Label protocol |
Fluorescently labelled target Cy3- and Cy5-cDNA was prepared from 250 ng of mRNA using a CyScribe First-Strand cDNA labelling kit (Amersham Biosciences) as per manufacture's instructions
|
| |
|
| Hybridization protocol |
1. Paired Cy3 and Cy5 labelled cDNA samples were combined and rotated in a Centrivap Concentrator (Labconco) at 35C until completely dried (approximately 30 minutes)
2. Samples were resuspended in 8 ul of nuclease free water (Amersham Biosciences) and denatured at 95C for 2 minutes before placing on ice for a minimum of 30 seconds
3. To reduce non-specific binding of target to the array surface, 2 uL of poly A (1 mg / 1 ml) was added to each sample and incubated at 75C for 45 minutes
4. Following the incubation, 10 ul of CyScribe kit hybrisation buffer and 20 ul of deionised formamide (Amresco) were added
5. The reaction mixtures were applied evenly across the array area of the prepared microarray slides, and coverslips were lowered with care, making sure no bubbles were present
6. Slides were placed into ArrayIt Hybridisation Cassettes (TeleChem International Inc) and 60 ul of 3 x SSC added to each well at the ends of the hybridisation cassettes
7. Slides were allowed to hybridise in the cassettes overnight in a 42C waterbath
|
| Scan protocol |
Hybridised microarray slides were scanned at 595 nm and 685 nm for detection of bound Cy3 and Cy5 cDNA respectively using the laser-induced fluorescence Agilent G2565BA Microarray Scanner System with SureScan Technology (Agilent Technologies). All slides were scanned using the default settings recommended by Agilent Technologies.
|
| Data processing |
Spot signal intensity data was computed from the microarray images using ImaGene 5.5 Standard Edition (Biodiscovery Inc.)
Analysis of the quantified image data was performed in the R statistical environment using LIMMA (Linear Models for Microarray Data). Microarray data was background corrected using the normexp method. An offset value of 50 was used during background correction to add a constant to the intensities before log-transforming so that log-ratios are shrunk towards zero at the lower intensities. Print-tip loess normalisation was performed for each microarray slide before Aquantile normalisation was applied to allow for comparisons between slides. Following normalisation, separate channel analysis was performed by fitting a linear model. For the assessment of differential expression an empirical Bayes method was used to moderate the standard errors for the estimated log-fold changes. This data has been analysed and is submitted for publication in the Journal of Reproduction, Fertility and Development. The study described in the manuscript focuses on gene expression changes between the time-points that are closest to each adjacent phase boundary. Therefore the normalised results submitted to GEO only represent the analysis of the following linear model contrasts: pregnancy day 26 versus lactation day 2 (phase 1 versus phase 2A), lactation day 110 versus lactation day 151 (phase 2A versus phase 2B) and lactation day 151 versus lactation 216 (phase 2B versus phase 3).
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| |
|
| Submission date |
Nov 26, 2014 |
| Last update date |
Nov 27, 2014 |
| Contact name |
Christy Vander Jagt |
| E-mail(s) |
christy.vanderjagt@depi.vic.gov.au
|
| Phone |
+61 3 90327212
|
| Organization name |
Department of Environment and Primary Industries
|
| Department |
Computational Biology
|
| Street address |
5 Ring Road, LaTrobe University
|
| City |
Bundoora |
| State/province |
Victoria |
| ZIP/Postal code |
3083 |
| Country |
Australia |
| |
|
| Platform ID |
GPL19466 |
| Series (1) |
| GSE63654 |
Gene expression in the mammary gland of the tammar wallaby during the lactation cycle |
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